Evidence for Rho protein regulation of renal tubular epithelial cell function  Robert J. Anderson, Carla J. Ray, Michel R. Popoff  Kidney International  Volume 58, Issue 5, Pages 1996-2006 (November 2000) DOI: 10.1111/j.1523-1755.2000.00372.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 Effect of Clostridium difficile toxin A on 3H-thymidine uptake (A) and migration, as assessed by rate of healing of small wounds made within confluent monolayers (B) in LLC-PK1 cells. For thymidine studies, each bar represents the mean ± SEM of four experiments done in triplicate under each condition. Values are expressed as a percentage of simultaneously studied controls. At 10 ng/mL and higher, Toxin A significantly (P < 0.001) decreased 3H-thymidine incorporation. For the migration experiments, each bar represents the mean ± SEM of four experiments. Values are expressed as a percentage of simultaneously studied controls. Symbols are: (▪) measurement at 6 hours; (?) measurement at 24 hours. Toxin A significantly (P < 0.05) decreased wound healing at each concentration and time point studied except at six hours for the 1.0 ng/mL dose (P = NS). Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 Effect of Clostridium difficile toxins A and B for 24 hours on LLC-PK1 apoptosis, as assessed by annexin V binding. Each bar represents the mean ± SEM of five experiments. Values are expressed as the percentage of cells demonstrating either necrosis (annexin V and propidium iodide uptake, ▪) or apoptosis (only annexin V uptake, (?)). A significant increase in apoptosis (P < 0.05) occurred with each tested concentration of Toxin A. At 0.1 ng/mL, Toxin A did not increase apoptosis (data not shown). At 100 ng/mL, Toxin B induced significant apoptosis (P < 0.025). Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Effect of C3 botulinum toxin for 24 hours on LLC-PK1 apoptosis and necrosis. The bars represent the mean ± SEM of four experiments. Values are expressed as the percentage of cells demonstrating either necrosis (▪) or apoptosis ((?)). At 1.0 μg/mL, C3 significantly (P < 0.02) increased both necrosis and apoptosis. Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of cytochalasin D on 3H-thymidine incorporation (A) and migration (B) in LLC-PK1 cells. In A, the bars represent the mean ± SEM of four experiments done in triplicate and are expressed as the percentage of simultaneously studied controls. At 0.1 and 1.0 μmol/L, cytochalasin D significantly decreased LLC-PK13H-thymidine uptake. In B, each bar represents the mean ± SEM of four experiments. Symbols are: (▪) measurements made six hours after wounding; (?) measurements made 24 hours after wound creation. At 0.1 and 1.0 μmol/L, cytochalasin D significantly (P < 0.01) impaired wound healing. Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 5 Effect of cytochalasin D for 24 hours (A) and (B) colchicine (▪) and control (lumicholchine; (?)) on LLC-PK1 apoptosis, as determined by annexin V binding. The bars represent the mean ± SEM of four experiments and express the percentage of cells with necrosis (?) or apoptosis (▪). At 1.0 and 10.0 μmol/L, cytochalasin D significantly (P < 0.001) increased apoptosis (A). At 1.0 and 10.0 μmol/L, colchicine slightly but significantly (P < 0.05) increased apoptosis (B). In data not shown, neither colchicine nor lumicholchine significantly affected the percentage of necrotic cells. Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 6 Effect of Clostridium difficile toxin A and cytochalasin D on LLC-PK1 expression of an antibody directed against Bcl-x. A representative immunoblot is shown. Lanes 1 and 3 represent solvent controls for toxin A (lane 2) and cytochalasin D (lane 4). Bcl-x has two isoforms: Bcl-xL with a molecular weight of 27 kD and Bcl-xs with a molecular weight of 19.5 kD. An effect of cytochalasin D to decrease expression of Bcl-xL is apparent. Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 7 Effect of Clostridium difficile toxin A and cytochalasin D (24 h) to affect LLC-PK1 expression of LLC-PK1 Bcl-xL. The bars represent the mean ± SEM of five experiments and depict the band area, as determined by densitometric analysis. Cytochalasin D modestly (P < 0.05) decreases expression of Bcl-xL. Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 8 Effect of a pancaspase inhibitor boc-aspartyl ([Ome]-fluoromethyl ketone) on Clostridium difficile toxin A-induced apoptosis (A) and cytochalasin D-induced apoptosis (B) in LLC-PK1 cells. The bars represent the mean ± SEM of four experiments and depict the percentage of apoptotic cells as assessed by annexin V binding. Symbols in A are: (▪) control cells; (?) toxin A-treated cells. Toxin A significantly (P < 0.01) induced apoptosis, and this apoptotic effect was abolished by a 100 μmol/L concentration of the pancaspase inhibitor. In B, the bars represent the mean ± SEM of five experiments and depict the percentage of cells with annexin V binding. Symbols are: (▪) control cells; (?) cytochalasin D-treated cells. At 100 μmol/L, the pancaspase inhibitor significantly (P < 0.05) reduced the effect of cytochalasin D zto induce apoptosis. Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 9 Effect of Clostridium difficile toxin A (100 ng/mL), lethal toxin 82 (1000 ng/mL), and cytochalasin D (10 μmol/L) on rhodamine phallotoxin staining of LLC-PK1 cells. (A) Control. (B) Toxin A. (C) Lethal toxin. (D) Cytochalasin D-treated cells. Toxin A and lethal toxin result in diffuse cytoplasmic redistribution, while cytochalasin D results in clumping of individual cells, diffuse cytoplasmic redistribution, and intracytoplasmic aggregation. Kidney International 2000 58, 1996-2006DOI: (10.1111/j.1523-1755.2000.00372.x) Copyright © 2000 International Society of Nephrology Terms and Conditions